Protein kinase C (also known as "calcium/phospholipid-dependent protein kinase", "PKC" or "C-kinase") is a family of closely related enzymes; one or more members of the protein kinase C family are found in nearly all animal tissues and animal cells that have been examined. The identity of protein kinase C is generally established by its ability to phosphorylate certain proteins when adenosine triphosphate and phospholipid cofactors are present, with greatly reduced activity when these cofactors are absent. Protein kinase C is believed to phosphorylate only serine and/or threonine residues in the proteins that are substrates for protein kinase C. Additionally, some forms of protein kinase C require the presence of calcium ions for maximal activity.
Protein kinase C comprises a family of ten or more closely related protein molecules [Parker, P. J. et al. Mol. Cell. Endocrin. 65: 1-11 (1989)]. Because of their high degree of relatedness they are referred to as "isozymes", "isotypes" or "isoforms". Occasionally the term "subtypes" is used, but this term is usually reserved to designate, as a subdivision, two or more variants of a single isotype.
The currently known isotypes of protein kinase C are: .alpha., .beta..sub.1, .beta..sub.2 and .gamma. (the "A-group"); .delta., .epsilon., .epsilon.' [Ono, Y. et al., J. Biol. Chem. 263: 6927-6932 (1988)], .eta. [also known as protein kinase C-L; Osada, S. et al., J. Biol. Chem. 265: 22434-22440 (1990) and Bacher, N. et al., Mol. Cell. Biol. 11: 126-133 (1991), respectively], and .theta. [Osada, S.-I. et al., Mol. Cell. Biol. 12: 3930-3938 (1992) (the "B-group"); .zeta. [Ono, Y. et al., J. Biol. Chem. 263: 6927-6932 (1988)] and .iota. [Selbie, L. A. et al., J. Biol. Chem. 268: 24296-24302 (1993), the latter also being known as PKC.lambda. [Akimoto, K. et al., J. Biol. Chem. 269: 12677-12683 (1994)] (the "C-group"); and .mu. [Johannes, F.-J. et al., J. Biol. Chem. 269: 6140-6148 (1994)], also known as PKD [Valverde, A. M. et al., Proc. Natl. Acad. Sci USA 91: 8572-8576 (1994) (the "D-group"). Members of the A-group require calcium ions for maximal activation, whereas the B-, C- and D-group members are thought to be largely calcium-independent for activation. The genes for each of the isotypes above have been cloned from one or more animal and yeast species and the clones have been sequenced; the relatedness of the genes and their product polypeptides is thus well established.
Beyond the ability of phospholipid and, for some isotypes, calcium to stimulate protein kinase C activity, members of the A-, B- and D-groups of the protein kinase C family are also substantially stimulated by certain 1,2-sn-diacylglycerols that bind specifically and stoichiometrically to a recognition site or sites on the enzyme. This site is called the diacylglycerol binding site, and it is located on the amino-terminal portion of protein kinase C, the so-called "regulatory domain". The carboxy-terminal portion of protein kinase C carries the site at which protein phosphorylation is effected, and this portion is therefore called the "kinase domain".
Thus, the rate at which various protein kinase C family members carry out their enzymatic phosphorylation of certain substrates can be markedly enhanced by the presence of the cofactors such as phospholipids, diacylglycerols (except for the C-group) and, for some protein kinase C family members, calcium ions. This stimulation of protein kinase C activity is referred to as protein kinase C "activation", and the activation of protein kinase C by the binding of diacylglycerols to the regulatory domain of protein kinase C is of particular importance in the normal and pathological functions of protein kinase C.
In contrast to the activation of protein kinase C, some chemical compounds have been shown, when added to protein kinase C enzyme assays, to reduce the rate at which protein kinase C phosphorylates its substrates; such compounds are referred to as protein kinase C "inhibitors" or, in some cases, "antagonists". In some circumstances, protein kinase C inhibitors are capable of inhibiting various cellular or tissue phenomena which are thought to be mediated by protein kinase C.
Activation of protein kinase C by diacylglycerols has been shown to be an important physiological event that mediates the actions of a wide variety of hormones, neurotransmitters, and other biological control factors such as histamine, vasopressin, .alpha.-adrenergic agonists, dopamine agonists, muscarinic cholinergic agonists, platelet activating factor, cytokines, growth factors and many others [see Y. Nishizuka, Nature 308: 693-698 (1984) and Science 225: 1365-1370 (1984) for reviews].
The biological role of protein kinase C is also of great interest because of the discovery that certain very powerful tumor promoting chemicals activate this enzyme by binding specifically and with very high affinity to the diacylglycerol binding site on the enzyme. In addition to diacylglycerols, there are at present six other known classes of compounds that bind to this site: diterpenes such as the phorbol esters; indole alkaloids (indolactams) such as the teleocidins, lyngbyatoxin, and indolactam V; polyacetates such as the aplysiatoxins and oscillatoxins; certain derivatives of diaminobenzyl alcohol; macrocyclic lactones of the bryostatin class; and, benzolactams such as (-)-BL-V8-310; these seven classes of compounds are collectively referred to herein as "phorboids". The phorbol esters have long been known as powerful tumor promoters, the teleocidins, aplysiatoxins, diacylglycerols and, though weak, the bryostatins are now known to have this activity, and it appears likely that additional classes of compounds will be found to have the toxic and tumor promoting activities associated with the capability to bind to the diacylglycerol site of protein kinase C and thus activate the enzyme. Other toxicities of these agents when administered to animals include lung injury and profound changes in blood elements, such as leukopenia and neuropenia, among many others.
Representative examples of the diterpene class of previously known protein kinase C-activating compounds are depicted below: ##STR1##
It can be seen that the phorboids depicted have diverse structural elements of both hydrophilic and hydrophobic nature, with one prominent exception, namely that each contains a hydroxymethyl or 1-hydroxyethyl group (indicated by the dashed-line boxes in each structure). Among the seven classes the diterpenes, indolactams, polyacetates, bryostatins and benzolactams have members of especially high potency, in the range of low nanomolar affinities for protein kinase C.
In addition to potent tumor promoting activity, these seven classes of compounds display a vast range of biological activities, as would be expected from the widespread distribution of their target enzyme. Many of these activities indicate the involvement of protein kinase C in important normal or pathological processes in animals, as shown by experiments utilizing both genetic and pharmacological approaches. Thus, the phorboids are potent skin inflammatory agents, cause smooth muscle contraction in several tissues, alter immune system function and can be used to cause or mimic a wide variety of other normal or pathological biological responses. Published evidence indicates that disease states such as the development of cancer, the onset and/or maintenance of inflammatory disease, the role of vasoconstriction in hypertension, the role of bronchoconstriction in asthma, the life cycles of many pathogenic human viruses, and the role of certain classes of cholinergic, adrenergic, serotoninergic and dopaminergic synapses in diseases of the central/peripheral nervous systems, may be mediated in vivo by the stimulation of protein kinase C or other diacylglycerol binding site-bearing entities by diacylglycerols, the latter being generated in the cell by either normal physiological or by pathological agents or conditions.
In analyzing the activity of a pharmaceutical or other bioactive compound, it is useful to consider two properties: the efficacy of the compound, defined as the capability to elicit a full or partial biological result, such as complete displacement of a ligand from its receptor site or the complete inhibition of inflammation or edema caused by a standard stimulus; and the potency, defined as that amount or concentration of drug that causes 50% of the full response (often abbreviated as the ED.sub.50). It is frequently the case within a given class of pharmaceutical agents that individual members of the class all have equal efficacy, i.e. they each can generate a full biological effect, but they show differing potencies. Thus, the structural modifications within such a class generally affect only the amount necessary to achieve a given result (i.e. the potency), and the modified compounds otherwise generally retain the same central biological characteristic (i.e. efficacy as a pharmaceutical or in a given biological assay or test). There may also be differences between members of such a class as regards properties other than the central biological characteristic; for example, members of the class might differ in side effects or susceptibility to metabolism by an organism.
Well-known pharmaceuticals that have been in extensive use for years or decades show a wide range of optimal therapueutic potencies. Aspirin, for example, is often taken in multi-gram amounts per day for treatment of inflammation or arthritis, and detailed analyses of its mechanism of action in vitro show that a concentration in the millimolar range is required for certain therapeutic effects of aspirin. In contrast, steroid-based topical anti-inflammatory compounds such as fluocinolone acetonide are many thousand-fold more potent, and, beyond this, some oral contraceptive agents are prescribed in daily doses in the microgram range. Thus, although high potency is generally advantageous for a pharmaceutical, it is not an absolute requirement.
The concepts of potency and efficacy provide a useful basis for understanding the properties of the nearly one thousand analogs of the typically skin-inflammatory and tumor-promoting phorboids that have been reported in the literature, including numerous examples on which major or minor chemical modifications have been made [see Evans and Soper, Lloydia 41: 193-233 (1978) and references cited therein]. When the structures of these phorboids are compared, and their activities for inflammation, tumor promotion and protein kinase C modulation are analyzed from the perspective of efficacy and potency, a remarkable unity is observed. To a degree that is nearly unique among known ligand-receptor phenomena, the structures of the different classes of phorboids vary quite markedly from one to the other class yet widespread testing of their biological activities has shown that these classes have essentially the same target site, namely the diacylglycerol binding site on protein kinase C, and generally have very similar biological properties. In particular, the numerous known phorboids of the diterpene, indolactam, diacylglycerol, polyacetate, bryostatin and benzolactam classes appear to have, with very minor exceptions, virtually identical efficacies as skin irritants and tumor promoters [T. Sugimura, Gann, 73: 499-507 (1982)]. Typical exceptions involve: (i) a few compounds that have a short duration of irritant activity and/or manifest diminished tumor promoting activity, perhaps due to slightly different protein kinase C isotype selectivity, toxicity or secondary parameters such as differing metabolic destruction rates; (ii) the bryostatins, which, though having weak but detectable inflammatory and tumor-promoting activity, show other, non-correlating pharmacological properties; or (iii) certain short-chain diesters of phorbol which are tumor promotion inhibitors at low doses but fully efficacious tumor promoters when tested alone at higher concentrations.
In contrast to the essentially equal efficacies among the vast majority of phorboids, their relative potencies cover a wide range, as measured in inflammation and promotion tests and as measured in numerous other in vivo and in vitro systems. Example compounds can be found in the diterpene, indolactam, polyacetate and benzolactam classes that have nearly equal, very high potencies. At the same time there are compounds in each of these classes which embody significant structural changes that do not diminish efficacy but do result in potency decreases of 10-fold to 100,000-fold or more [see, for example, Driedger and Blumberg, Cancer Res. 37: 3257-3265 (1977), Cancer Res. 39: 714-719 (1979)]. Thus, all these compounds appear to be capable of achieving generally the same biological results, and merely differ in the amount which must be used to obtain a given result.
In vitro measurements of biochemical properties provide an even more sensitive method for comparing the properties of the various phorboids. For example, using a radioactively labeled phorboid such as [.sup.3 H]phorbol 12,13-dibutyrate or [.sup.3 H]lyngbyatoxin, one can measure the potency of a test compound as a competitive ligand for the diacylglycerol binding site, which is also referred to herein as the "phorboid binding site" on protein kinase C or on other biological molecules which have phorboid binding sites (see below). Alternatively, one can measure the ability of a given phorboid to stimulate the protein kinase C-mediated incorporation of radioactive phosphate from [.sup.32 P]adenosine triphosphate into a standard acceptor substrate such as histone H1. These tests reveal a difference in potency between given phorboid agonists of as much as 10,000,000-fold or more [Dunn and Blumberg, Cancer Res. 43: 4632-4637 (1983), Table 1].
These basic data regarding the phorboid agonists are an important consideration because they underscore the concept that the structural differences among the phorboids known prior to the instant invention, especially the widely studied diterpenes, indolactams, diacylglycerols, polyacetates and bryostatins, generally do not affect their efficacies as toxic agonists, and indeed a remarkably wide variety of structural changes are tolerated in this regard. Such changes generally alter potency only and do not provide agents with therapeutic utility, since the resulting compounds retain their toxicity.
Some minor changes in phorboid structure are known to result in generally inactive compounds, such as a stereochemical change from 4-.beta. to 4-.alpha. in the phorbol series, and indeed some of the diterpene skeleton structures carry hydroxy groups that must be esterified in order for inflammatory activity to be observed. However, these inactive compounds are quite few in number among the known phorboids, and no therapeutic utility has been demonstrated for them.
The phorbol esters, indolactams, polyacetates, diaminobenzyl alcohols, and bryostatins are generally found in plants, molds, and algae, or are synthetic in origin. Although they are found in many parts of the world, normal human contact with these classes of phorboids is thought to be low and of negligible medical significance. In contrast, the diacylglycerols are part of the functioning of virtually every type of animal cell, and the undesirable activation (and, alternatively as discussed below, the cessation of desirable activation) of protein kinase C by the diacylglycerols is thought to have a very widespread role in human diseases.
Thus, compounds capable of blocking the activation of, or inhibiting, protein kinase C by acting as specific pharmacological antagonists of the diacylglycerols at the diacylglycerol binding site on protein kinase C, would be valuable agents in the prevention and treatment of a wide variety of diseases in animals and humans. For example, the need for, and potential utility of, protein kinase C inhibitors/antagonists as agents for the treatment of cancer has received much attention [D. Corda, et al., Trends in Pharmacological Sciences 11: 471-473 (1990); G. Powis, Trends in Pharmacological Sciences 12: 188-194 (1991); S. Gandy and P. Greengard, Trends in Pharmacological Sciences 13: 108-113 (1992); B. Henderson and S. Blake, Trends in Pharmacological Sciences 13: 145-152 (1992)].
It is possible that the different protein kinase C isozymes have different biological roles, and published evidence supports this idea [Homan, E., Jensen, D. and Sando, J., J. Biol. Chem. 266: 5676-5681 (1991); Gusovsky, F. and Gutkind, S., Mol. Pharm. 39: 124-129 (1991); Borner, C., "The Role of protein kinase C in Growth Control", Sixth International Symposium on Cellular Endocrinology, W. Alton Jones Cell Science Center, Lake Placid, N.Y., Aug. 12-15, 1990; Naor, Z. et al., Proc. Natl. Acad. Sci. USA 86: 4501-4504 (1989); Godson, C., Weiss, B. and Insel, P., J. Biol. Chem. 265: 8369-8372 (1990); Melloni, E. et al., Proc. Natl. Acad. Sci. USA 87: 4417-4420 (1990); Koretzky, G. et al., J. Immunology 143: 1692-1695 (1989)]. For example, the stimulation of one protein kinase C isotype or a limited subset of protein kinase C isotypes might lead to undesirable results such as the development of inflammation [Ohuchi, K. et al., Biochim. Biophys. Acta 925: 156-163 (1987)], the promotion of tumor formation [Slaga, T., Envir. Health Perspec. 50: 3-14 (1983)] or an increased rate of viral replication in cells (i.e., de novo infection of cells and/or expression, assembly and release of new viral particles) [Harada, S. et al., Virology 154: 249-258 (1986)].
On the other hand, other protein kinase C isozymes might be responsible for the many beneficial effects observed when protein kinase C is stimulated by known protein kinase C activators in a variety of biological settings; such beneficial effects include the cessation of division of leukemic cells [Rovera, G., O'Brien, T. and Diamond, L., Science 204: 868-870 (1979)], multiplication of colonies of lymphocytes [Rosenstreich, D. and Mizel, S., J. Immunol. 123: 1749-1754 (1979)] and leucocytes [Skinnider, L. and McAskill, J., Exp. Hematol. 8: 477-483 (1980)] or the secretion of useful bioregulatory factors such as interferon-.gamma. [Braude, I., U.S. Pat. No. 4,376,822] and interleukin-2 [Gillis, S., U.S. Pat. No. 4,401,756].
Recent publications indicate that diacylglycerol binding sites exist on newly-described proteins which lack the kinase domain, and thus lack the kinase activity, of protein kinase C. One such protein is n-chimaerin, found in human brain [Ahmed et al., Biochem. J. 272: 767-773 (1990)] and the other is the unc-13 gene product of the nematode Caenorhabditis elegans, [Maruyama, I. and Brenner, S., Proc. Natl. Acad. Sci. USA 88: 5729-5733 (1991)]. The presence of the diacylglycerol binding sites on these two proteins was demonstrated by standard binding experiments with [.sup.3 H]phorbol 12,13-dibutyrate. These new proteins may have other enzymatic or biological activities which can be modulated by compounds which bind to their diacylglycerol binding sites. Thus, such compounds may have utility on non-protein kinase C biological targets.
Given that there are now numerous distinct biological entities bearing diacylglycerol binding sites, it would be highly desirable to obtain chemical compounds which could specifically and selectively target one or another type of diacylglycerol binding site, thus permitting one to selectively activate or inhibit one such site without affecting the others. Such compounds would be valuable experimental tools for studying the role of individual types of proteins bearing diacylglycerol binding sites as well as providing novel means for treating diseases in which protein kinase C or other diacylglycerol binding site-bearing proteins are involved.
There are several published reports describing chemical compounds capable of selectively distinguishing several diacylglycerol/phorboid-type binding sites in mouse skin [Dunn and Blumberg, op. cit.] and in purified preparations of protein kinase C isotypes [Ryves, W. J., et al., FEBS Letters 288: 5-9(1991)]. However, in these studies, even the compounds showing the clearest differences in affinity for these distinct classes, namely phorbol 12,13-dibutyrate, 12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-phenylacetate and thymeleatoxin, are only selective by a factor of 10-1000 in dissociation constant among the different binding sites. Furthermore, these compounds have potent skin inflammatory activity and are not desirable in human or animal medicine because of this toxicity.
Thus, to briefly recapitulate, two kinds of new compounds relating to diacylglycerol binding sites would be highly desirable. The first type would be capable of selectively activating one or a few useful, but not other, deleterious, diacylglycerol binding site-bearing targets. The second type would be capable of inhibiting, or antagonizing the stimulation of, one or more diacylglycerol binding site-bearing targets without blocking activity and/or activation of phorboid-activated target entities whose activation is physiologically harmless or desirable. These kinds of compounds would be valuable agents for the study of diacylglycerol binding site-bearing entities and for the prevention or treatment of a wide range of human and animal diseases thought to involve protein kinase C or other entities under the control of diacylglycerol binding sites.
The physiology of protein kinase C includes, in certain cases, a phenomenon known as "down-regulation", manifested as the ability of protein kinase C activators of the phorboid class to initially stimulate protein kinase C at or shortly after the time of application of the phorboid, followed by a net, phorboid-induced metabolic lowering of total protein kinase C levels. [See e.g. Cooper, D. R. et al., Biochem. Biophys. Res. Comm. 161: 327-334 (1989); Isakov, N. et al., J. Biol. Chem. 265: 2091-2097 (1990); Strulovici, B. et al., J. Biol. Chem. 266: 168-173 (1991); and Gschwendt, M. et al., FEBS Letters 307: 151-155 (1992). Several of these studies illustrate the selective down-regulation of one but not another protein kinase C isotype via extended exposure to standard protein kinase C-activating phorboids such as phorbol esters.] Thus, at short times the phorboids generally act as protein kinase C stimulants, but at longer times, for certain protein kinase C isotypes in certain biological settings, the net loss of one or more protein kinase C isotypes in response to the (initially stimulatory) phorboid results in substantial or complete loss of the protein kinase C. This important effect is therefore functionally equivalent to inhibition of protein kinase C, even though the effect is achieved by nominal activators of this enzyme family, and it is clear that, on a long-term time scale, net inhibition of protein kinase C can be achieved either with inhibitors or, in many cases, activators of protein kinase C. A given compound might therefore show quite complex properties on protein kinase C; for example, a non-toxic agonist might fail to stimulate the protein kinase C isotype(s) responsible for inflammation while at the same time activating many other isotypes on a short time scale and inhibiting a subset of the latter isotypes on a long time scale.
With the exception of clinical tests of bryostatin 1 itself [Prendiville, J. et al., Brit. J. Cancer 68: 418-424 (1993)] and of certain modifications to the hydroxymethyl/1-hydroxyethyl group present in all currently known phorboids [P. E. Driedger and J. Quick, U.S. Pat. No. 5,145,842 and related patents and patent applications], efforts to make medical use of the previously known phorboids themselves or to modify the structures of these known phorboids in medically useful ways, have generally not been successful in producing useful compounds with toxicity low enough for use in humans.
For example, it has been known for some time that several of the toxic, inflammatory and tumor-promoting compounds such as phorbol 12-tigliate 13-decanoate, mezerein, lynobyatoxin and aplysiatoxin have anti-leukemic activity in mouse model tests [T. Sugimura, op cit.; S. M. Kupchan and R. L. Baxter, Science 187: 652-653 (1975); S. M. Kupchan, et al., Science 191: 571-572 (1976); M. C. Territo and H. P. Koeffler, Br. J. Haematol. 47, 479-483 (1981)]. However, these compounds are all extremely toxic to many tissues and are cancer suspect agents, making them quite unattractive for consideration as human therapeutic agents.
Ganong, et al. [Proc. Nat. Acad. Sci. USA 83: 1184-1188 (1986)] tested a series of diacylglycerols and found no antagonistic activity in that series against the standard agonist, 1,2-dioctanoylglycerol. Compounds tested in this work were modified in the hydroxymethyl or other portions of the diacylglycerol molecule, and these modifications produced only a loss of activity or a weakened activity that was not distinguishable from the agonist activity of 1,2-dioctanoylglycerol itself, a compound which is toxic to mouse skin [R. Smart, et al., Carcinogenesis 7: 1865-1870 (1986); A. Verma, Cancer Res. 48: 2168-2173 (1988)]. These modified diacylglycerols were not antagonists in these tests and no utility was found. Schmidt and Hecker ("Simple phorbol esters as inhibitors of tumor promotion by TPA in mouse skin". Carcinogenesis, Vol. 7, ed. by E. Hecker et al., Raven Press, New York, 1982, pp. 57-63) studied the abilities of a series of diterpene phorboids to inhibit tumor promotion by the standard phorboid agonist tumor promoter phorbol 12-myristate 13-acetate (PMA, also known as TPA). They found that, at low doses, some short-chain ester derivatives of phorbol, differing from PMA only in the chain length of the 12- and 13-ester substituents were able to block the tumor promotion by PMA. However, all of the compounds that were active as antagonists at low doses are also known to be very efficacious skin irritants themselves at slightly higher doses and most of them are also known to have tumor promoting activity. Thus, these short-chain esters still have toxic inflammatory and tumor promoting activity at doses only slightly different from those which would be needed to exhibit a therapeutic effect in mice. In the same study (Schmidt and Hecker, op. cit.) phorbol 12-myristate (abbreviated "TP" in the Schmidt and Hecker publication), which differs from PMA only in the lack of a substituent on the 13-hydroxy group, was tested as an inhibitor of tumor promotion and was found to be inactive.